The sputtering properties of two representative cluster ion beams in secondary ion mass spectrometry (SIMS),
Au3+, have been directly compared. Organic thin films consisting of trehalose and dipalmitoylphosphatidylcholine (DPPC) are employed as prototypical targets. The strategy is to make direct comparison of the response of a molecular solid to each type of the bombarding cluster by overlapping the two ion beams onto the same area of the sample surface. The ion beams alternately erode the sample while keeping the same projectile for spectral acquisition. The results from these experiments are important to further optimize the use of cluster projectiles for SIMS molecular depth profiling experiments. For example,
Au3+ bombardment is found to induce more chemical damage as well as Au implantation when compared with
C60+ is found to be able to remove the damage and the implanted Au effectively. Discussions are also presented on strategies of enhancing sensitivity for imaging applications with cluster SIMS.
The use of large cluster primary ions (e.g. C60, Au400) in secondary ion mass spectrometry has become prevalent in recent years due to their enhanced emission of secondary ions, in particular, molecular ions (MW ≤ 1500 Da). The co-emission of electrons with SIs was investigated per projectile impact. It has been found that SI and electrons yields increased with increasing projectile energy and size. The use of the emitted electrons from impacts of C60 for localization has been demonstrated for cholesterol deposited on a copper grid. The instrumentation, methodologies, and results from these experiments are presented.
Electron Emission Microscope; Cluster SIMS; Electron Emission; Localization
This paper describes the application of nanoparticle bombardment with time-of-flight secondary ion mass spectrometry (NP-ToF-SIMS) for the analysis of native biological surfaces for the case of sagittal sections of mammalian brain tissue. The use of high energy, single nanoparticle impacts (e.g. 520 keV Au400) permits desorption of intact lipid molecular ions, with enhanced molecular ion yield and reduced fragmentation. When coupled with complementary molecular ion fragmentation and exact mass measurement analysis, high energy nanoparticle probes (e.g. 520 keV Au400 NP) provide a powerful tool for the analysis of the lipid components from native brain sections without the need for surface preparation and with ultimate spatial resolution limited to the desorption volume per impact (~103 nm3).
Molecular depth profiling of organic overlayers was performed using a mass selected C60 ion beam in conjunction with time-of-flight (TOF-SIMS) mass spectrometry. The characteristics of sputter depth profiles acquired for a 300-nm Trehalose film on silicon were studied as a function of the kinetic impact energy of the projectile ions. The results are interpreted in terms of a simple model describing the balance between sputter erosion and ion induced chemical damage. It is shown that the efficiency of the projectile to clean up the fragmentation debris produced by its own impact represents a key parameter governing the success of molecular depth profile analysis.
Fundamental advances in secondary ion mass spectrometry (SIMS) now allow for the examination and characterization of lipids directly from biological materials. The successful application of SIMS-based imaging in the investigation of lipids directly from tissue and cells are demonstrated. Common complications and technical pitfalls are discussed. In this review, we examine the use of cluster ion sources and cryogenically compatible sample handling for improved ion yields and to expand the application potential of SIMS. Methodological improvements, including pre-treating the sample to improve ion yields and protocol development for 3-dimensional analyses (i.e. molecular depth profiling), are also included in this discussion. New high performance SIMS instruments showcasing the most advanced instrumental developments, including tandem MS capabilities and continuous ion beam compatibility, are described and the future direction for SIMS in lipid imaging is evaluated.
ToF-SIMS; lipids; cluster sources; sample preparation; C60+QSTAR; J105 3D Chemical Imager and imaging mass spectrometry (IMS)
Time-of-flight Secondary ion mass spectrometry (ToF-SIMS) provides a method for the detection of native and exogenous compounds in biological samples on a cellular scale. Through the development of novel ion beams the amount of molecular signal available from the sample surface has been increased. Through the introduction of polyatomic ion beams, particularly C60, ToF-SIMS can now be used to monitor molecular signals as a function of depth as the sample is eroded thus proving the ability to generate 3D molecular images. Here we describe how this new capability has led to the development of novel instrumentation for 3D molecular imaging while also highlighting the importance of sample preparation and discuss the challenges that still need to be overcome to maximise the impact of the technique.
Sputter depth profiling of organic films while maintaining the molecular integrity of the sample has long been deemed impossible because of the accumulation of ion bombardment-induced chemical damage. Only recently, it was found that this problem can be greatly reduced if cluster ion beams are used for sputter erosion. For organic samples, carbon cluster ions appear to be particularly well suited for such a task. Analysis of available data reveals that a projectile appears to be more effective as the number of carbon atoms in the cluster is increased, leaving fullerene ions as the most promising candidates to date. Using a commercially available, highly focused C60q+ cluster ion beam, we demonstrate the versatility of the technique for depth profiling various organic films deposited on a silicon substrate and elucidate the dependence of the results on properties such as projectile ion impact energy and angle, and sample temperature. Moreover, examples are shown where the technique is applied to organic multilayer structures in order to investigate the depth resolution across film-film interfaces. These model experiments allow collection of valuable information on how cluster impact molecular depth profiling works and how to understand and optimize the depth resolution achieved using this technique.
Molecular depth profiling; Cluster SIMS; Carbon clusters; Cluster ion beams
Salts play a mysterious role in desorption mass spectrometry, especially in biological samples. We used trehalose films doped with a peptide as a well defined model system to investigate the ionization effects in organic molecular depth profiling. Sodium salts at 1% level were added into the solution used to produce the trehalose films, and depth profiles were obtained with a C60 ion source. The results show that the protonated molecular ion signal from the peptide and the quasimolecular ion signal of trehalose are significantly suppressed by the addition of salts, whereas the signals representing salt clusters and salt adducts of trehalose are formed in both positive and negative modes. The formation of protonated molecular ions is found to correlate with the ratio between protonated and bare water ions, suggesting that the latter can be used as an indicator for the accumulation of protons liberated by the ion bombardment. In experiments where no salt was added, it is shown that the surface variation of the protonated molecular ion signal strongly depends upon the water content of the trehalose film.
molecular depth profiling; SIMS; C60; trehalose film; ionization; cluster ions
This paper describes the advantages of using single impacts of large cluster projectiles (e.g. C60 and Au400) for surface mapping and characterization. The analysis of co-emitted time-resolved photon spectra, electron distributions and characteristic secondary ions shows that they can be used as surface fingerprints for target composition, morphology and structure. Photon, electron and secondary ion emission increases with the projectile cluster size and energy. The observed, high abundant secondary ion emission makes cluster projectiles good candidates for surface mapping of atomic and fragment ions (e.g., yield >1 per nominal mass) and molecular ions (e.g., few tens of percent in the 500 < m/z < 1500 range).
photon emission; electron emission; secondary ion yield; cluster-SIMS
This paper describes a new technique for fabrication of nanostructured porous silicon (pSi) for laser desorption ionization mass spectrometry. Porous silicon nanowell arrays were prepared by argon plasma etching through an alumina mask. Porous silicon prepared in this way proved to be an excellent substrate for desorption/ionization on silicon (DIOS) mass spectrometry (MS) using adenosine, Pro-Leu-Gly tripeptide and [Des-Arg9]-bradykinin as the model compounds. It also allows the analyses of complex biological samples such as a tryptic digest of bovine serum albumin, and a carnitine standard mixture. Nanowell array surfaces were also used for direct quantification of the illicit drug fentanyl in red blood cell extracts. This method also allows full control of the surface features. MS results suggested that the pore depth has significant effect on the ion signals. Significant improvement in the ionization was observed by increasing the pore depth from 10 nm to 50 nm. These substrates are useful for laser desorption ionization in both the atmospheric pressure and vacuum regimes.
The need of cellular and sub-cellular spatial resolution in LDI / MALDI Imaging Mass Spectrometry (IMS) necessitates micron and sub-micron laser spot sizes at biologically relevant sensitivities, introducing significant challenges for MS technology. To this end we have developed a transmission geometry vacuum ion source that allows the laser beam to irradiate the back side of the sample. This arrangement obviates the mechanical / ion optic complications in the source by completely separating the optical lens and ion optic structures. We have experimentally demonstrated the viability of transmission geometry MALDI MS for imaging biological tissues and cells with sub-cellular spatial resolution. Furthermore, we demonstrate that in conjunction with new sample preparation protocols, the sensitivity of this instrument is sufficient to obtain molecular images at sub-micron spatial resolution.
Imaging Mass Spectrometry; sub-cellular; sub-micron spatial resolution; transmission geometry; single cell imaging
Mass spectrometry imaging and profiling of individual cells and subcellular structures provide unique analytical capabilities for biological and biomedical research, including determination of the biochemical heterogeneity of cellular populations and intracellular localization of pharmaceuticals. Two mass spectrometry technologies—secondary ion mass spectrometry (SIMS) and matrix assisted laser desorption ionization mass spectrometry (MALDI MS)—are most often used in micro-bioanalytical investigations. Recent advances in ion probe technologies have increased the dynamic range and sensitivity of analyte detection by SIMS, allowing two- and three-dimensional localization of analytes in a variety of cells. SIMS operating in the mass spectrometry imaging (MSI) mode can routinely reach spatial resolutions at the submicron level; therefore, it is frequently used in studies of the chemical composition of subcellular structures. MALDI MS offers a large mass range and high sensitivity of analyte detection. It has been successfully applied in a variety of single-cell and organelle profiling studies. Innovative instrumentation such as scanning microprobe MALDI and mass microscope spectrometers enable new subcellular MSI measurements. Other approaches for MS-based chemical imaging and profiling include those based on near-field laser ablation and inductively-coupled plasma MS analysis, which offer complementary capabilities for subcellular chemical imaging and profiling.
secondary ion mass spectrometry; matrix assisted laser desorption/ionization; subcellular profiling; elemental imaging; mass cytometry; tissue imaging
Photoionization of molecules sputtered from molecular thin films has been achieved using high field 125 fs pulses in the mid-IR spectral range. Using several model systems, we show that it is possible to significantly reduce molecular fragmentation induced by the laser field by increasing the photoionization wavelength. By examining the photoionization spectra as a function of wavelength, it is apparent that the photoionization mechanism is changing from a non-adiabatic multi-electron excitation process to a process that involves tunnel ionization. The results of these observations are discussed in terms of their significance for bioimaging with focused ion beams and mass-spectrometry.
Integrating surface plasmon resonance analysis with mass spectrometry allows detection and characterization of molecular interactions to be complemented with identification of interaction partners. We have developed a procedure for Biacore®3000 that automatically performs all steps from ligand fishing and recovery to sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry including on-target digestion. In the model system used in this study a signal transduction protein, calmodulin, was selectively captured from brain extract by one of its interaction partners immobilized on a sensor chip. The bound material was eluted, deposited directly onto a MALDI target, and analyzed by mass spectrometry both as an intact protein and after on-target tryptic digestion. The procedure with direct deposition of recovered material on the MALDI target reduces sample losses and, in combination with automatic sample processing, increases the throughput of surface plasmon resonance mass spectrometry analysis.
Surface plasmon resonance; biomolecular interaction assay; mass spectrometry; ligand fishing; protein complexes
Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 μm wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 μm and ~40 μm, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.
Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is capable of determining the distribution of hundreds of molecules at once directly from tissue sections. Since tissues are analyzed intact without homogenization, spatial relationships of molecules are preserved. The technology is, therefore, undoubtedly powerful to investigate the molecular complexity of biological processes. However, several technical refinements are essential for full exploitation of MALDI-IMS to dictate dynamics alteration of biomolecules in situ; these include ways to collect tissues, target-specific tissue pretreatment, matrix choice for efficient ionization, and matrix deposition method to improve imaging resolution. Furthermore, for MALDI-IMS to reach its full potential, quantitative property in the IMS should be strengthened. We review the challenges and new approaches for optimal imaging of proteins, lipids and metabolites, highlighting a novel quantitative IMS of energy metabolites in the recent literature.
MALDI; imaging mass spectrometry; CE-MS; tissue-rinsing
A new approach is described for imaging mass spectrometry analysis of drugs and metabolites in tissue using matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR). The technique utilizes the high resolving power to produce images from thousands of ions measured during a single mass spectrometry (MS)-mode experiment. Accurate mass measurement provides molecular specificity for the ion images on the basis of elemental composition. Final structural confirmation of the targeted compound is made from accurate mass fragment ions generated in an external quadrupole-collision cell. The ability to image many small molecules in a single measurement with high specificity is a significant improvement over existing MS/MS based technologies. Example images are shown for olanzapine in kidney and liver and imatinib in glioma.
Carbon cluster emission from thin carbon foils (5–40 nm) impacted by individual Aun+q cluster projectiles (95–125 qkeV, n/q = 3–200) reveals features regarding the energy deposition, projectile range, and projectile fate in matter as a function of the projectile characteristics. For the first time, the secondary ion emission from thin foils has been monitored simultaneously in both forward and backward emission directions. The projectile range and depth of emission were examined as a function of projectile size, energy, and target thickness. A key finding is that the massive cluster impact develops very differently from that of a small polyatomic projectile. The range of the 125 qkeV Au100q+q (q ≈ 4) projectile is estimated to be 20 nm (well beyond the range of an equal velocity Au+) and projectile disintegration occurs at the exit of even a 5 nm thick foil.
gold nanoparticles; thin foils; carbon clusters; projectile range; secondary ion mass spectrometry
In the present work, the advantages of a new, 100kV platform equipped with a massive gold cluster source for the analysis of native biological surfaces are shown. Inspection of the molecular ion emission as a function of projectile size demonstrate a secondary ion yield increase of ~100x for 520 keV Au400+4 as compared to 130 keV Au3+1 and 43 keV C60. In particular, yields of tens of percent of molecular ions per projectile impact for the most abundant components can be observed with the 520 keV Au400+4 probe, respectively. A comparison between 520 keV Au400+4 ToF-SIMS and MALDI-MS data showed a similar pattern and similar relative intensities of lipids’ components across a rat brain sagittal section. The abundant secondary ion yields of analyte-specific ions makes 520 keV Au4004+ projectiles an attractive probe for sub-μm molecular mapping of native surfaces.
secondary ion yield; molecular ion emission; cluster-SIMS
Molecular depth profiling and three-dimensional imaging using cluster projectiles and SIMS have become a prominent tool for organic and biological materials characterization. To further explore the fundamental features of cluster bombardment of organic materials, especially depth resolution and differential sputtering, we have developed a reproducible and robust model system consisting of Langmuir–Blodgett (LB) multilayer films. Molecular depth profiles were acquired, using a 40-keV C60+ probe, with LB films chemically alternating between barium arachidate and barium dimyristoyl phosphatidate. The chemical structures were successfully resolved as a function of depth. The molecular ion signals were better preserved when the experiment was performed under cryogenic conditions than at room temperature. A novel method was used to convert the scale of fluence into depth which facilitated quantitative measurement of the interface width. Furthermore, the LB films were imaged as a function of depth. The reconstruction of the SIMS images correctly represented the original chemical structure of the film. It also provided useful information about interface mixing and edge effects during sputtering.
Molecular depth profiling; 3D imaging; Langmuir; Blodgett films; Multilayers; Cluster beam; SIMS
A new method of performing Mössbauer spectroscopy using a fast shutter in combination with microfocused synchrotron radiation is demonstrated.
A new method of performing Mössbauer spectroscopy with synchrotron radiation is demonstrated that involves using a high-speed periodic shutter near the focal spot of a microfocused X-ray beam. This fast microshuttering technique operates without a high-resolution monochromator and has the potential to produce much higher signal rates. It also offers orders of magnitude more suppression of unwanted electronic charge scattering. Measurement results are shown that prove the principle of the method and improvements are discussed to deliver a very pure beam of Mössbauer photons (E/ΔE ≃ 1012) with previously unavailable spectral brightness. Such a source will allow both Mössbauer spectroscopy in the energy domain with the many advantageous characteristics of synchrotron radiation and new opportunities for measurements using X-rays with ultra-high energy resolution.
Mössbauer spectroscopy; nuclear resonant scattering; X-ray shutter
The current limitation for SIMS analyses is insufficient secondary ion yields, due in part to the inefficiency of traditional primary ions. Massive gold clusters are shown to be a route to significant gains in secondary ion yields relative to other commonly used projectiles. At an impact energy of 520 keV, Au400+4 is capable of generating an average of greater than ten secondary ions per projectile, with some impact events generating >100 secondary ions. The capability of this projectile for signal enhancement is further displayed through the observation of up to seven deprotonated molecular ions from a single impact on a neat target of the model pentapeptide leu-enkephalin. Positive and negative spectra of leu-enkephalin reveal two distinct emission regimes responsible for the emission of either intact molecular ions with low internal energies or small fragment species. The internal energy distribution for this projectile is measured using a series of benzylpyridinium salts and compared with the small polyatomic projectile Au3+ at 110 keV as well as distributions previously reported for electrospray ionization and fast atom bombardment. These results show that Au400+4 offers high secondary ion yields not only for small fragment ions, e.g. CN–, typically observed in SIMS analyses, but also for characteristic molecular ions. For the leu-enkephalin example, the yields for each of these species are greater than unity.
Cluster-SIMS; ion yield; internal energy; peptide fragmentation; leu-enkephalin; angiotensin
Ambient ionization imaging mass spectrometry is uniquely suited for detailed spatially-resolved chemical characterization of biological samples in their native environment. However, the spatial resolution attainable using existing approaches is limited by the ion transfer efficiency from the ionization region into the mass spectrometer. Here we present a first study of ambient imaging of biological samples using nanospray desorption ionization (nano-DESI). Nano-DESI is a new ambient pressure ionization technique that uses minute amounts of solvent confined between two capillaries comprising the nano-DESI probe and the solid analyte for controlled desorption of molecules present on the substrate followed by ionization through self-aspirating nanospray. We demonstrate highly sensitive spatially resolved analysis of tissue samples without sample preparation. Our first proof-of-principle experiments indicate the potential of nano-DESI for ambient imaging with a spatial resolution of better than 12 μm. The significant improvement of the spatial resolution offered by nano-DESI imaging combined with high detection efficiency will enable new imaging mass spectrometry applications in clinical diagnostics, drug discovery, molecular biology, and biochemistry.
imaging mass spectrometry; nanospray desorption electrospray ionization (nano-DESI); rhodamine; tissue; spatial resolution
In recent years, the deluge of complicated molecular and cellular microscopic images creates compelling challenges for the image computing community. There has been an increasing focus on developing novel image processing, data mining, database and visualization techniques to extract, compare, search and manage the biological knowledge in these data-intensive problems. This emerging new area of bioinformatics can be called ‘bioimage informatics’. This article reviews the advances of this field from several aspects, including applications, key techniques, available tools and resources. Application examples such as high-throughput/high-content phenotyping and atlas building for model organisms demonstrate the importance of bioimage informatics. The essential techniques to the success of these applications, such as bioimage feature identification, segmentation and tracking, registration, annotation, mining, image data management and visualization, are further summarized, along with a brief overview of the available bioimage databases, analysis tools and other resources.
Supplementary information: Supplementary data are available at Bioinformatics online.
A C60+ cluster ion projectile is employed for sputter cleaning biological surfaces to reveal spatio-chemical information obscured by contamination overlayers. This protocol is used as a supplemental sample preparation method for time of flight secondary ion mass spectrometry (ToF-SIMS) imaging of frozen and freeze-dried biological materials. Following the removal of nanometers of material from the surface using sputter cleaning, a frozen-patterned cholesterol film and a freeze-dried tissue sample were analyzed using ToF-SIMS imaging. In both experiments, the chemical information was maintained after the sputter dose, due to the minimal chemical damage caused by C60+ bombardment. The damage to the surface produced by freeze-drying the tissue sample was found to have a greater effect on the loss of cholesterol signal than the sputter-induced damage. In addition to maintaining the chemical information, sputtering is not found to alter the spatial distribution of molecules on the surface. This approach removes artifacts that might obscure the surface chemistry of the sample and are common to many biological sample preparation schemes for ToF-SIMS imaging.